A major objective of this project is to study the biochemical properties of tRNA molecules with defined structural modifications. The co-crystal structure of E. coli tRNA(Gln) with its cognate synthetase will be used as a guide to preparing modified tRNAs with single atomic substitutions. The affinity of the modified tRNA with the enzyme will be measured using filter binding, fluorescence and enzymatic assays. The overall goal is to understand the relationship of the crystal structure to the interaction in solution and to understand the energetics of the interaction. The large changes in the structure of tRNA upon protein binding are particularly interesting. The proposed allosteric linkage between the anticodon and the active site will be investigated. The interaction of EF-Tu with tRNA will be studied in further detail. The contribution of the phosphodiester backbone of tRNA to protein binding will be accessed by preparing modified backbones. Derivatives of a minimal aminoacyl tRNA substrate for EF-Tu which emerged from an in vitro selection experiment will be further analyzed. An in vitro selection strategy is proposed to isolate RNAs that mimic the ribosome by stimulating the hydrolysis of GTP by EF-Tu. Finally, in vitro selection experiments with glutamine tRNA synthetase are proposed to isolate RNAs that fold like tRNA, but have very different sequences. We believe that tRNA-like folding motifs are present in many cellular RNAs, but are unrecognized because their sequence requirements are not known.